**2.2 Nutrient usage and fermentation performance**

The right use of metabolites is key for a successful fermentation. One of the most important steps in the fermentation process is the hexose uptake. Overexpression of fructose/H<sup>+</sup> symporter *FSY1* from *S. pastorianus* results in improve glucose and fructose uptake during wine fermentation [25]. Moreover, using a null hexose transporter mutant *HXT1* to *HXT7* of *S. cerevisiae* (KOY.TM6\*P) and overexpression of chimeric *HXT1*-*HXT7* gene in this strain showed that there is a decreased ethanol production and increased biomass under high glucose concentration [8]. The first step of the glycolysis is depend on the role of cytosolic thioredoxins 1 and 2. The double mutant of these thioredoxins in the haploid wine yeast C9 (derived from commercial strain L2056) has a problem in the use of the sugars at the levels of the hexokinase 1 and 2 and in the glucokinase 1 that produce a slow fermentation [26].

One of the most important nutrients in the grape juice is the nitrogen and it could be a limiting nutrient for the growth of yeast because low levels of nitrogen can stop the fermentation when the sugars are still remained in the medium. *S. cerevisiae* cannot assimilate inorganic nitrogen nor polypeptides and proteins, so its grow depend on ammonium and free amino acids, called YAN (Yeast Assimilable Nitrogen). Concentrations below 140 mg/L of YAN in a normal sugar concentration, can produce negative effect in the fermentation process and nitrogen depletion irreversibly arrest hexose transport. One way to improve the nitrogen assimilation is through deletion of *URE2* repressor of alternative nitrogen sources as prolines. It controls the *PUT1*-encoded proline oxidase and *PUT2*-encoded pyrroline-5-carboxylate dehydrogenase to create yeast that can efficiently assimilate the abundant supply of proline and arginine in grape juice [25, 27]. *MFA2* deletion (encoding mating factor-a) is another way to improve the fermentation efficiency under nitrogen limitation (75 mg/L). They used a deletion in the haploid wine yeast AWRI1631 under microvinification conditions [28]. Another work by Jin Zahng using a transposon library in wine yeast, selected five candidate genes to efficiently complete a model of oenological fermentation with limited nitrogen availability. They did the gene disruptions in the haploid wine yeast C911D where they found that the deletion of *ECM33* (GPI-anchored protein involved in efficient glucose uptake) resulted in the shortest fermentation (up to 31%) in grape juice and there were no differences in the nitrogen utilization, cell viability or biomass with the parental strain. This mutant has an up-regulation in the cell way integrity regulated genes [29].

### **2.3 Increasing the quality of the wine**

Understanding wine flavor compound composition is a key to improve the final product. Yeast metabolism during wine fermentation produce ethanol and secondary metabolites that are important for the wine. The generation of wine yeast able to produce wines with reduced ethanol concentrations while retaining harmonious balance between the level of alcohol, acidity, sweetness, and other sensory qualities has been the focus of extensive research. The main idea is to divert partially the carbon metabolism from the formation of ethanol to glycerol, but it is difficult to do it without a significant impact on wine quality, as acetic acid rises [30]. For example, overexpression of the main glycerol producing enzyme *GPD1* (NAD-dependent glycerol-3-phosphate dehydrogenase) together with the deletion of *ALD6* (aldehyde dehydrogenase) is able to decrease acetic acid production in the strain AWRI2531 and produce a fermentation with 15–20% less ethanol and more glycerol [31]. Reduction of 7.4% of ethanol without negative consequences was possible through the partial deletion of *PDC2* (transcription factor required for expression of the two isoforms of pyruvate decarboxylase *PDC1* and *PDC5*) [32]. Overexpression of *TPS1* (trehalose synthase gene) produce a 10% ethanol decrease [33]. NADH oxidase was expressed in *S. cerevisiae* so the NADH pool was reduce getting a 15% lower of ethanol but the redox reactions and grow was affected [34]. Decreases in ethanol levels was carry on by expression of *GOX1* (glucose oxidase gene) from *Aspergiullus niger* [35]. Alternative, deletion of TORC1 pathway kinase *SCH9* in the haploid wine yeast C9, increase glycerol production during wine making conditions [36].

The most significant effect on the aroma of wine are acetate esters, ethyl acetate (fruity and tart aromas), 2-phenylethyl acetate (honey, rose) and isoamyl acetate (banana flavor) [37]. Increase these compounds in the wine is important to get a good final product. Overexpression of *ATF1* (alcohol acetyltransferase) got a significant increase in acetate ester production. Moreover, deletion of *ATF1* and *ATF2* abolished the formation of isoamylacetate but still produces ethyl acetate and overexpression of esterase (*IAH1*) decrease significantly concentration of ethyl acetate and isoamyl acetate among others [38, 39].

Terpenoids or isoprenoids are naturally compounds which are involved in the fragrance and aroma of flowers and fruits. One way to improve the production of these positive compounds in the wine is using genes from species that produce this aroma. For example, using S-linalool synthase (*LIS*) from *Clarkia breweri* in *S. cerevisae* produce a novo production of linalool in wine about 19 μg/L [40]. Through the expression of the *Ocimun basilicum* (sweet basil) geraniol synthase (*GES*) gene in the industrial wine yeast T73, Pardo *et al.* got an recombinant yeast which excreted geraniol de novo at an amount 750 μg/L that was further metabolized in other interested monoterpenoids and esters as citronellol, linalool, nerol, citronellyl acetate and geranyl acetate [41]. Expression and secretion of the *Aspergillus awamori* α-Larabinofuranoside in combination with either β-glucosidase from *Saccharomycopsis fibuligera* or from *Aspergiluus kawachii* in the industrial yeast VIN13 has higher concentrations of monoterpenoids and improve sensory characteristics [42].

Other volatile sulfur compound is hydrogen sulfur, H₂S, that has an undesirable 'sulfurous', 'rotten egg'-like off flavor even at low concentrations (1 μg/L) that it is a significant problem for the global wine industry. Reduced H₂S amount in the wine it is another improvement that can has beneficial effects for the wine. Specific site directed mutation in both *MET10* and *MET5* genes (α and β subunits of sulfite reductase enzyme) reduced by 50–99% the H₂S production depending on the strain [43]. Using the strain UCD932 a strain producing little or no detectable H₂S during wine fermentation was constructed and identified the allele of *MET10* (*MET10–932*) as a responsible. Replacing the *MET10* allele of high- H₂S producing strain with *MET10–932* prevented H₂S formation [44].

### **2.4 Improving human health**

Yeast metabolism can be diverted to produce compounds that has specific influence in human health. This section will focus on two beneficial compounds for human health (resveratrol and hydroxytyrosol) and one potentially dangerous, ethyl carbamate.

Grape juice has a lot of polyphenols, one of them, resveratrol is a stress metabolite produced by *Vitis vinifera* grape vines and it is a potent antioxidant with multiple beneficial effects. Red wines contain a much higher resveratrol concentration than white wine, due to skin contact during fermentation [45]. In plants, resveratrol synthesis is from malonyl-CoA and *p*-coumaroyl-CoA by the resveratrol synthase. But *in S. cerevisiae* coenzyme -A ligase is absent, and it is necessary for the last steps of the resveratrol synthesis. In 2003, Becker *et al.* by co-expressing the coenzyme-A ligase gene (*4CL216*) from a hybrid polar and the grapevine resveratrol synthase gene (*vstl1*) resveratrol production was successfully for the first time. Introduction of 4 heterologous genes (phenylalanine ammonia lyase gene from *Rhodosporidium toruloides*, the cinnamic acid 4-hydroxylase and 4-coumarate coenzyme A ligase genes both from *Arabidopsis thaliana*, and the stilbene synthetase gene from *Arachis hypogaea*), overexpression of acetyl-CoA carboxylase gene (*ACC1*) and addition of tyrosine to the medium produced an increase in concentration of resveratrol up to 5.8 mg/L in *S. cerevisiae* laboratory W303-1A strain [46]. Moreover, two expression vector carrying 4-coumarate coenzyme A ligase gene (4CL) from *Arabidopsis thaliana* and resveratrol synthase gene (RS) from *Vitis vitifera* were introduced in the industrial yeast EC1118 [47]. This strain produced 8.25 mg/L of resveratrol. Indeed, resveratrol was produced with fed-batch fermentation directly from glucose (416.65 mg/L) and from ethanol (531.41 mg/L) [48]. With an optimization of the same strategy with the electron transfer to the cytochrome P450 monooxygenase,

800 mg/L of resveratrol was obtained [49]. Recently, a co-culture platform with two different species was used to produced 36 mg/L [50]. *Escherichia coli* excrete *p*-coumaric acid into the media and *S. cerevisiae* with an inactivation-resistant version of acetyl-CoA carboxylase (ACC1S659A,S1157A) that modulate constitutively the expression of 4-coumarate-CoA ligase from *Arabidopsis thaliana* (4CL) and resveratrol synthase from *Vitis vinifera* (STS) to produce resveratrol.

Another polyphenol that has a strong antioxidant capacity is hydroxytyrosol (HT). It is found it in extra virgin olive oil, less in wine (with a range between 0.28–9.6 mg/L). In yeast, tyrosol is synthesized from tyrosine through the wellestablished Ehrlich pathway. In bacteria, there are some ways to produce hydroxytyrosol using yeast genes. For example, co-expression of yeast *ARO8* and *ARO10* genes for an important accumulation of tyrosol when was added in the media. Moreover, co-expression of yeast *ARO10* and *ADH6* and the overexpression of the native aromatic hydroxylase complex HpaBC produce important amounts (647 mg/L) of HT in *E. coli* [51]. Recently, HT production (4 mg/L) was possible with the introduction of the *E. coli* hydroxylase HpaBC complex components (hpaB and hpaC) in laboratory BY4743 yeast strain and with the addition of tyrosol to the media [52].

Ethyl Carbamate (EC) is a toxic present in wines. During wine fermentation, *S. cerevisiae* metabolizes arginine (one of the major amino acid in grape juice) using arginase *CAR1* to ornithine and urea, but this urea is not fully metabolizing and is secreted. Urea degradation is an energy-dependent two-step process catalyzed by urea amidolyase (*DUR1*, *2* genes). The urea that is secreted by yeast to the media can react with the ethanol of the wine to form ethyl carbamate that is classified as probably carcinogenic for humans. With the overexpression of *DUR1* and *DUR2* under *PGK1* promoter, 89.1% less of EC was developed in Chardonnay wine [53]. Deletion of *CAR1* in the YZ22 strain blocked urea secretion and there is a reduction of EC production [54]. This fermentation results showed that the content of urea and EC in wine decreased by 77.89% and 73.78% respectively and no differences were detected in growth and fermentation parameters with the parental strain.
